Immunoassays for determining vitamin B12, and reagents and kits therefor

Abstract

Methods are described for the development of monoclonal antibodies to intrinsic factor and the application of said antibodies in the development of B12 immunoassays. In particular, antibodies are described that are capable of binding to intrinsic factor only in the absence of vitamin B12, and that are released from binding in the presence, and upon the binding, of vitamin B12 to intrinsic factor. Assays may be formatted such that either antibody or intrinsic factor is immobilized or labeled.

1 . A composition useful for determining the presence of vitamin B12 in a sample, the composition comprising the antibody produced from the hybridoma deposited at the American Type Culture Collection and assigned ATCC number ATCC-HB10711.  
   
   
       2 - 4 . (canceled)  
   
   
       5 . A kit for assaying for vitamin B12 in a sample comprising (a) a predetermined amount of a labeled antibody that is capable of specifically binding to intrinsic factor only in the absence of vitamin B12, and that is released from binding in the presence, and upon the binding, of vitamin B12 to intrinsic factor, and (b) a predetermined amount of an intrinsic factor bound to a solid phase support.  
   
   
       6 - 8 . (canceled)  
   
   
       9 . A diagnostic assay method for vitamin B12 in a liquid sample comprising: 
 (a) contacting the sample with a known amount of intrinsic factor bound to a solid phase and a known amount of a labeled antibody which specifically binds to intrinsic factor in an amount related to the presence or amount of vitamin B12 in the sample, said antibody being capable of binding to a site on intrinsic factor that is distinct from the site on intrinsic factor to which vitamin B12 binds and which binds to intrinsic factor only in the absence of vitamin B12, and that is released from binding in the presence, wherein the intrinsic factor will specifically bind to vitamin B12 in the sample to form a vitamin B12-intrinsic factor complex;    (b) separating the vitamin B12-intrinsic factor complex; and    (c) determining the amount of vitamin B12 by measuring the amount of label associated with the vitamin B12-intrinsic factor complex.    
   
   
       10 . (canceled)